Blood coagulation is a process consisting of a complex interaction of various blood components, i.e., factors, which eventually gives rise to a fibrin clot. Generally, the blood components which participate in what has been referred to as the coagulation "cascade" are proenzymes or zymogens, i.e., enzymatically inactive proteins which are converted to proteolytic enzymes by the action of an activator which is, itself, an activated clotting factor. Coagulation factors that have undergone such a conversion are generally referred to as "active .alpha. factors," and are designated by the addition of a lower case "a" suffix (e.g., Factor VIIa).
Activated Factor X ("Xa") is required to convert prothrombin to thrombin, which then converts fibrinogen to fibrin as a final stage in forming a fibrin clot. There are two systems, i.e., pathways, that promote the activation of Factor X. The "intrinsic pathway" refers to those reactions that lead to thrombin formation through utilization of factors present only in plasma. A series of protease-mediated reactions ultimately generates Factor IXa which, in conjunction with Factor VIIIa, cleaves Factor X into Xa. An identical proteolysis is effected by Factor VIIa and its co-factor, tissue factor, in the "extrinsic pathway" of blood coagulation. Tissue factor is a membrane bound protein and does not normally circulate in plasma. Upon vessel disruption, however, it can complex with Factor VII or Factor VIIa to catalyze Factor X activation or Factor IX activation in the presence of Ca.sup.2+ and phospholipid. While the relative importance of the two coagulation pathways in hemostasis is unclear, Factors IX activation by the Factor VIIa-tissue factor complex has, in recent years, been found to play a pivotal role in the initiation of the normal clotting response. However, Factor IX activation in response to tissue factor exposed at sites of vascular injury has also been implicated in thrombosis, a pathological manifestation of the clotting cascade in blood vessels.
Thrombosis, which can complicate rupture of an atherosclerotic plague, can cause partial or total occlusion of the affected blood vessel, thereby leading to a number of important cardiovascular complications, including unstable angina, acute myocardial infarction (heart attack), or cerebral vascular accidents (stroke). Vessel injury and/or stasis can trigger venous thrombosis causing deep vein thrombosis and subsequent pulmonary embolism. Such diseases are a major cause of disability and mortality throughout the world, but particularly in Western societies. Moreover, thrombin and, in particular, surface-bound thrombin plays a role in thrombus formation in cardiac bypass circuits, after angioplasty and during and after thrombolytic therapy for acute myocardial infarction. Therefore, patients undergoing these procedures must be treated with very high doses of anticoagulants or antithrombin agents. Although high doses of these agents may effectively prevent clotting, they can give rise to serious bleeding complications.
The clot or thrombus, which forms as a result of activation of the clotting cascade, contains fibrin, platelets and numerous other blood components. Thrombin bound to fibrin remains active and causes growth of the clot by continued cleavage of fibrinogen and activation of platelets and other coagulation factors, such as Factor V and factor VIII. Moreover, unlike free thrombin which is readily inactivated by naturally occurring anti-thrombins (e.g., antithrombin III (ATIII)), clot-bound thrombin is protected from inactivation. As a result, the clot acts as a reservoir for active thrombin which triggers further clot growth. In addition, thrombin also induces smooth cell proliferation and, thus, may be involved in proliferative responses, such as graft-induced atherosclerosis and restenosis after angioplasty or atherectomy.
Because thrombin is critical to thrombus formation, the use of thrombin inhibitors for treating thrombosis and thrombotic complications has long been proposed. A number of partially effective inhibitors have been in use for years. Heparin, for example, can be used as an anticoagulant and antithrombin agent to inhibit fibrin formation, platelet aggregation and thrombus formation. Heparin, however, has a number of limitations. For example, it has biophysical limitations because it acts as an anticoagulant by activating ATIII and, thus, it is relatively ineffective at inactivating fibrin-bound thrombin at safe doses, thereby allowing the continued growth of thrombus mediated by thrombin bound to fibrin in the pre-existing thrombus. In addition, the doses required to produce an antithrombotic effect are quite unpredictable and, therefore, the dosage must be monitored closely. Low molecular weight heparins (LMWHs) can also be used as anticoagulants and anti-thrombin agents to inhibit fibrin formation, platelet aggregation and thrombus formation. LMWHs act by activating ATIII and, as such, have the same biophysical limitations as heparin. However, LMWHs produce a more predictable anticoagulant effect than heparin. Thus, both heparin and LMWH have the limitation of not readily inactivating surface-bound thrombin. The consequences of this are (a) high concentrations are needed to achieve an anti-thrombin effect which can lead to excessive bleeding, and (b) once the agents are cleared from the circulation, the surface-bound thrombin can reactivate clotting.
Inactivation of clot-bound thrombin may be achieved with another set of compounds known as direct thrombin inhibitors. Such inhibitors include hirudin and its derivatives, and inhibitors of the active site of thrombin, such as argatroban and PPACK (D-phenylalanyl-L-propyl-L-arginyl chloromethyl ketone). Hirudin is an anti-thrombin substance extracted from the salivary glands of leeches. Related compounds include hirulog which is a small, synthetic analog of hirudin. While these drugs are able to inhibit clot-bound thrombin, they have the following limitations. First, they do not typically inactivate clot-bound thrombin selectively, but do so at the same concentrations which are required to inhibit free thrombin. Secondly, their inactivation of thrombin is generally stoichiometric and, thus, unless very high concentrations are used, the inhibitory effect can be overcome by the large amounts of thrombin that are generated at sites where surface-bound thrombin accumulates (e.g., on bypass circuits, or at sites of arterial or venous thrombosis). As a result of the above two limitations, high concentrations of direct thrombin inhibitors (e.g., hirudin) must typically be administered to interact with and inhibit the free thrombin generated by clot-bound thrombin. Such high inhibitor concentrations can, however, cause unwanted bleeding. Moreover, direct thrombin inhibitors (e.g., hirudin, its analogs and small molecule active site thrombin inhibitors, such as argatroban) are generally reversible and, thus, their inhibitory effect is lost when the drugs are cleared from the blood. Unfortunately, this reversible inhibition can lead to rebound activation of coagulation.
In addition, inactivation of clot-bound thrombin may be achieved with a third class of compounds which bind reversibly or irreversibly to the active, i.e., catalytic, site of thrombin. PPACK is an example of an irreversible active site inhibitor. Such inhibitors, however, generally lack sufficient specificity for thrombin and, thus, have questionable safety. For example, PPACK also inactivates tissue type plasminogen activator, the major initiator of clot lysis. Thus, PPACK administration could trigger thrombosis by blocking the major pathway for clot degradation. Moreover, inhibitors such as PPACK have the same limitation as hirudin in that they typically have equal activity against clot-bound and free thrombin. This is problematic because evidence indicates that total inhibition of free thrombin using irreversible active site inhibitors may lead to excessive bleeding.
Moreover, other anticoagulant and antithrombin agents have been described in the literature. For instance, hirudin derivatives for blocking the active site of thrombin are described in U.S. Pat. Nos. 5,240,913 and 5,196,404. A bifunctional anti-thrombotic composition which includes both a glycoprotein IIb/IIIa inhibitory domain and a thrombin inhibitory domain is described in WO 92/10575. Peptide analogs of glycoprotein IIIa for thrombogenesis inhibition are described in WO 90/00178. Inhibitors of factor X and/or Xa are described in U.S. Pat. Nos. 5,239,058 and 5,189,019, and PCT publications WO 93/09803, WO 92/04378 and WO 92/01464. Inhibitors of factors VII and/or VIII are described in U.S. Pat. Nos. 5,223,427 and 5,023,236 and WO 92/06711. Platelet anti-adhesives and related antibodies are described in WO 92/08472. For a review of the structure and function of thrombin, see, Stubbs and Bode, Thrombosis Research 69:1-58 (1993).
In addition, numerous modified heparin compositions, as well as other glycosaminoglycans and their derivatives, have been developed. For example, U.S. Pat. Nos. 5,296,471, 5,280,016 and 5,314,876 describe the desulfation of heparin, periodate oxidation of heparin/heparan sulfates followed by reduction of resulting aldehyde groups, and high molecular mass N,O-sulfated heparosans, respectively. Low molecular weight heparin fractions have been used for several years (see, Boneu, et al., Thrombosis Research 40:81-89 (1985)). More recently, various dermatan sulfates have been developed and their interactions with heparin cofactor II studied (see, Mascellani, et al., Thrombosis Research 74:605-615 (1994), and Sheehan, et al., J. Biol. Chem. 289:32747-32751 (1994)). For a review of the limitations of heparin and the potential advantages of new anticoagulants as anti-thrombotics, see, Hirsh, Circ. 88:I-C (1993).
In view of the foregoing, there remains a need in the art for improved compositions and methods that are useful, for example, for inhibiting thrombogenesis associated with cardiovascular disease. An ideal antithrombotic agent would be one which can pacify the clot by inactivating fibrin-bound thrombin at concentrations which do not produce abnormal bleeding resulting from inhibition of thrombin production in the general circulation and/or which can selectively block the coagulation cascade at a desirable point. The present invention fulfills these and other needs.